Iron (Fe) is a major micronutrient which influences plant growth, development, quality and yield. Although basic helix-loop-helix (bHLH) transcription factors (TFs) which respond to iron deficiency have been identified, the molecular mechanisms have not been fully elucidated. In this study, a novel bHLH TF, NtbHLH1, was found to be induced by iron deficiency. Further analysis indicated that NtbHLH1 is localized to the nucleus and functions as a transcriptional activator. Moreover, overexpression of NtbHLH1 resulted in longer roots, altered rhizosphere pH and increased ferric-chelate reductase activity in iron deficient conditions. Overall these changes resulted in increased iron uptake relative to wild type plants. NtbHLH1 mutants, on the other hand, had an opposite phenotype. In addition, transcript levels of seven genes associated with iron deficiency response were higher in the NtbHLH1 overexpression transgenic plants and lower in ntbhlh1 relative to the WT under iron deficiency treatment. Taken together, these results demonstrated that NtbHLH1 plays a key role in iron deficiency response and they provide new insights into the molecular basis of iron homeostasis in tobacco. (C) 2019 Elsevier Inc. All rights reserved.